First-in-Human Brain Imaging of [18F]TRACK, a PET tracer for

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Letter

First-in-Human Brain Imaging of [18F]TRACK, a PET tracer for Tropomyosin Receptor Kinases Justin J. Bailey, Lena Kaiser, Simon Klaus Lindner, Melinda Wuest, Alexander Thiel, JeanPaul Soucy, Pedro Rosa-Neto, Peter J. H. Scott, Marcus Unterrainer, David R. Kaplan, Carmen Wängler, Björn Wängler, Peter Bartenstein, Vadim Bernard-Gauthier, and Ralf Schirrmacher ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00144 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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ACS Chemical Neuroscience

First-in-Human Brain Imaging of [18F]TRACK, a PET tracer for Tropomyosin Receptor Kinases

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§

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Justin J. Bailey,† Lena Kaiser, Simon Lindner, Melinda Wüst†, Alexander Thiel‡,∩, Jean-Paul Soucy‡,

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Pedro Rosa-Netoꝼ, Peter J.H. Scottꝼ, Marcus Unterrainer , David R. Kaplan╪, Carmen Wängler♦, Björn

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Wängler⌠, Peter Bartenstein, Vadim Bernard-Gauthier † and Ralf Schirrmacher†,

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†Department

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§

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‡

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∩Jewish

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ꝼTranslational Neuroimaging Laboratory, McGill Centre for Studies in Aging, Douglas Mental Health University Institute, Montreal, QC H4H 1R3, Canada

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ꝼDivision

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╪Program

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♦Biomedical

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⌠Molecular

of Oncology, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

Department of Nuclear Medicine, Ludwig-Maximilians-University of Munich, Munich 81377, Germany

McConnel Brain Imaging Centre, Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Quebec H3A 2B4, Canada General Hospital, Lady Davis Institute, Montreal, QC HT3 1E2, Canada

of Nuclear Medicine, Department of Radiology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON M5G 0A4,

Canada Chemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, 68167 Mannheim, Germany Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Mannheim 68167, Germany

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Author contributions: P.B., P. R. N., V.B.-G., A.T., C. W., B. W., D.R. C., R.S. designed research; J.J.B., M.W., S.L., L.K., B.W., P.B., V.B.-G., M.U., J.P.S., R.S. performed research; P.B., R.S. contributed new reagents/analytical tools; J.J.B., M.W., S.L., L.K., B.W., P.B., V.B.-G., R.S. analyzed data; J.J.B., V.B.-G., L.K, R.S wrote the paper.

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Corresponding

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Email: [email protected], Phone: 780-248-1829

Author:

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ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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V.B.-G.

and R.S. contributed equally to this work.

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2 ACS Paragon Plus Environment

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ABSTRACT

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The tropomyosin receptor kinase TrkA/B/C family is responsible for human neuronal growth, survival and

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differentiation from early nervous system development stages onward. Downregulation of TrkA/B/C

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receptors characterizes numerous neurological disorders including Alzheimer disease (AD). Abnormally

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expressed Trk receptors or chimeric Trk fusion proteins are also well-characterized oncogenic drivers in a

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variety of neurogenic and non-neurogenic human neoplasms and are currently the focus of intensive clinical

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research. Previously, we have described the clinical translation of a highly selective and potent carbon-11-

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labeled pan-Trk radioligand and the preclinical characterization of the optimized fluorine-18-labeled analog,

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[18F]TRACK, for in vivo Trk PET imaging. We describe herein central nervous system selectivity

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assessment and first-in-human study of [18F]TRACK.



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KEYWORDS

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Tropomyosin receptor kinase, Trk, positron emission tomography, PET, neuroimaging, fluorine-18, copper-

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mediated radiofluorination, kinase inhibitor.

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ACS Paragon Plus Environment

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The tropomyosin receptor kinases A, B, and C (TrkA, TrkB, and TrkC) are single-pass transmembrane

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tyrosine kinase receptors encoded by the NTRK1, NTRK2, and NTRK3 genes respectively, which are

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primarily expressed within the peripheral (PNS) and central nervous systems (CNS) in humans where they

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sustain neuronal survival and regulate synaptic maturation and plasticity.1-3 Trk kinases bind the

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neurotrophins (nerve growth factor (NGF); brain-derived neurotrophic factor (BDNF); neotrophin-3 (NT-3);

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and neurotrophin-4 (NT-4)), which elicit receptor dimerization/trans-autophosphorylation and subsequent

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Ras/MAPK, PI3K/AKT and PLCγ/PKC pathway activation.1-4 As a consequence, Trk dysregulation is often

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associated with neurological disorders, neurodegenerative diseases and cancer.5-7 For example, changes

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in the expression level or dysfunctional Trk signaling are early molecular changes found in the progression

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from no cognitive impairment (NCI) to mild cognitive impairment (MCI) and MCI to Alzheimer’s disease

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(AD).8-10 Relevant to oncology, the TPM3-NTRK1 gene fusion (non-muscle tropomyosin) trk, from which

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the current name of the full-length Trk receptors normally expressed in neurons is derived, was one of the

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first oncogenic fusion products identified.12,13 Dozens of Trk fusion proteins, wherein a Trk kinase domain

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is juxtaposed to an unrelated sequence often undergoing spontaneous ligand-independent oligomerization,

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have been identified at various frequencies in human cancers.14 These chimeric proteins, which can

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hyperactivate the same prosurvival and proliferation signaling pathways as neurotrophin-induced activation

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of normal full-length neuronal Trk receptors, act as oncogenic drivers in multiple tumor types. Recently,

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these molecular targets were shown to be actionable in the clinic using novel Trk kinase inhibitors, most

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notably larotrectinib, under both histology- and age-agnostic paradigms (Figure 1).14,15

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We recently reported on Trk-targeted radioligands for PET imaging including the first-in-human

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assessment of [11C]-(R)-IPMICF16 ([11C]4) and the discovery and evaluation in non-human primates of the

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fluorine-18 derivative [18F]TRACK ([18F]5) (Figure 1).16-18 These first-in-class radiotracers for TrkB/C brain

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imaging were characterized and evaluated in four species, rat, mice, non-human primates and in case of

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[11C]4 also in humans. [11C]4 however showed a few disadvantages with regards to human in vivo imaging:

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Firstly, its

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state is not reached within that physical time frame. The objective of the current study was to analyze first-

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in-human data of [18F]TRACK as to its in vivo stability, brain penetration, kinetics and safety and directly

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compare its in vivo brain distribution and kinetics to [11C]4.

11C-label

does not allow for prolonged imaging protocols and secondly, the equilibrium binding



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[INSERT FIGURE 1]

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Detailed characterization obtained in the course of the development and preclinical evaluation of

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[18F]TRACK to date demonstrated that this radiotracer exhibits overall excellent properties to serve as a

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PET ligand for Trk, which include: 1) ease of one-step labeling with fluorine-18, 2) high on-target in vitro

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potency (4.21, 0.15, and 0.31 nM for human TrkA, TrkB, and TrkC), 3) excellent kinome selectivity as

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analyzed through a panel of 369 human kinases (no detectable kinase off-target at 1000-fold concentration

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over TrkB/C potency), and 4) specific and selective binding to TrkB/C in human brain tissue in vitro – all of

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which with TrkB/C constituting high density targets in the mammalian brain.17,19 The decision of moving

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from the short-lived carbon-11-labeled tracer [11C]-(R)-IPMICF16 to [18F]TRACK was also motivated by in

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vitro data indicating a significant reduction as a human P-glycoprotein/breast cancer resistance protein (P-

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gp/BCRP) efflux substrate which could be favorable with respect to brain uptake and retention of this new

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radiotracer.17 We previously demonstrated that [18F]TRACK engages a type I binding mode with the TrkA

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ATP binding site.17 Considering the possibility that our Trk inhibitor could display interactions with non-

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kinase targets,20,21 we first determined the detailed selectivity mapping of [19F]TRACK with respect to a

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diverse array of CNS-rich receptors and channels and other relevant drug targets. We found inhibitor

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[19F]TRACK to display no off-target interaction to any significant extent (no inhibition >30% of control

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specific binding) against the 80 proteins tested at concentration of 0.2 M, corresponding to ~1000-fold

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over TrkB/C affinity (Table 1). These data, taken with our previous kinome data, further demonstrate the

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specificity of our lead.

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[INSERT TABLE 1]

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[INSERT FIGURE 2]

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For the first-in-human imaging study, high molar activity [18F]TRACK (Am = 188 GBq /mol) was

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obtained as previously described from the corresponding phenylboronic acid pinacol ester precursor using

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a copper-mediated radiolabeling with [Cu(OTf)2(pyr)4] in DMA nBuOH media.17 The radioligand was shown


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to exhibit all required quality control prerequisites for human use including high radio-chemical and chemical

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purity (HPLC, GC), radionuclide purity (γ-spectrometry) and absence of endotoxins (Table S1). The

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neuroimaging study was performed in one healthy male subject (43 years of age). The production and

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administration was done in accordance to the German Medicinal Products act and ethical approval was

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obtained prior to the investigation of the human subject. There were no adverse events or clinically

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observable pharmacologic effects reported following intravenous administration of [18F]TRACK, during the

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scanning or post experiment. Plasma analysis of the unmetabolized fraction of [18F]TRACK is presented in

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Figure 2. We only observed a moderate decrease in the intact plasma fraction of [18F]TRACK over the

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course of the scan duration with ~80% remaining [18F]TRACK at 90 min post-injection. After bolus injection,

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the activity concentration during first pass of the radioligand in the human brain peaked at 30 s (flow related

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first pass). This was followed by an increasing uptake peaking at about 20 min p.i. and decreasing

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thereafter. Overall, the regional uptake was consistent with the known expression and distribution of TrkB/C

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(cerebellum ~ thalamus > cortex > white matter) and previous preclinical imaging data.16,17 The distribution

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was very similar to our previous carbon-11 lead, [11C]-(R)-IPMICF16.16 Contrary to [11C]-(R)-IPMICF16

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however, which reached a plateau with no apparent washout from the brain between 5-60 min, our

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tracer displayed faster and reversible kinetics throughout the 90 min scanning time. Figure 3 and Figure 4

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respectively show the SUV images of [18F]TRACK (including MR overlay) and the regional brain time activity

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curves (TACs) in comparison to [11C]-(R)-IPMICF16, both evaluated in the same human subject. The SUV-

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ratios based on white matter (WM, Table 2) were slightly higher between 10 and 60 minutes p.i. for the Trk

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expression-rich regions thalamus, cerebellum and cortex for [18F]TRACK compared to [11C]-(R)-IPMICF16,

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indicating more favorable imaging characteristics of the

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observable radioactivity uptake in the skull, we concluded that [18F]TRACK did not suffer from defluorination

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in vivo to any significant extent. The differences in brain kinetics between [18F]TRACK and our previous

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lead also align well with the ~ 6-fold higher affinity for [11C]-(R)-IPMICF16 compared to the tracer presented

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here and are consistent with kinetics found in rhesus monkey. We note that the brain uptake and kinetics

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of [18F]TRACK are highly consistent between mice (Figure S1), non-human primate17 and human subjects.

18F-labelled

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18F-

compound. Based on the lack of


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[INSERT TABLE 2]

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[INSERT FIGURE 3]

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Although the current study demonstrates that [18F]TRACK crosses the blood brain barrier and

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accumulates in accordance to the known TrkB/C expression and brain distribution, it is important to

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recognize that the brain uptake is only moderate which may hinder the ability to assess TrkB/C expression

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when they diminish during the course of disease, and is largely similar to [11C]-(R)-IPMICF16 despite

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different kinetics. This observation, when considered with the known difference in efflux pump liabilities

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between the two radiotracers, appears inconsistent with the hypothesis that P-gp/BCRP-mediated efflux is

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an important determinant to account for the limited uptake in human for both tracers. We speculate that

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other ATP-binding cassette transporters may play a role in [18F]TRACK brain efflux or the suboptimal

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pharmacokinetic (PK) parameters that are currently under research focus.

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[INSERT FIGURE 4]

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Besides the extensive first-in-human PET analysis for the uptake of [18F]TRACK into the different brain

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regions, additional dynamic PET experiments were carried out in mice. As analyzed from the human PET

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data uptake, washout and retention profiles of [18F]TRACK in the mouse brain as seen from the TACs were

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very similar to our previously studied carbon-11 radiotracer, [11C]-(R)-IPMICF16 (Figure S1), which may be

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justified by the almost identical chemical structure of both TrK radiotracers (Figure 1). This was also visible

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in very similar IC50 values against TrKA, TrKB and TrKC as determined in a [γ-33P]ATP-based enzymatic

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binding assay16, 17. Development of an 18F-labeled radiotracer allowed for longer PET imaging times which

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was and will be also of an advantage for translation into a patient setting thus allowing for more detailed

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analysis after background washout. Taken together the present mouse data supports the findings in the

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human brain, although with less detailed analysis from the different brain regions. The data from non-human

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primates are also in line with the current human data.


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Overall, our results show that [18F]TRACK exhibits high pan-Trk selectivity and can be efficiently

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synthesized in one radiochemical step, sufficient RCYs and high molar activity (Am) using Cu-catalyzed

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late-stage fluorination of non-activated aromatic systems. We further demonstrate that this radioligand is

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stable in human in vivo and can be safely administered for clinical research. Our translational data however

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indicate that despite apparent reduction in efflux liabilities (specifically towards the major efflux transport P-

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gp and BCRP) in model mammalian cell lines, the radioligand [18F]TRACK ([18F]5) suffers from similarly

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moderate brain uptake in human as observed with the previously analyzed [11C]-(R)-IPMICF16 ([11C]4).

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Determining whether this limitation is primarily due to contributions from other unaccounted for ATP-binding

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cassette transporters or suboptimal PK parameters will require additional structural refinement. A clinical

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study of [18F]5 in control subjects, Alzheimer Disease and Mild Cognitive Impairment patients is currently

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planned and will be able to answer these questions.

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METHODS

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Material and General Methods.

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CNS Target Binding Assays. Inhibitor [19F]TRACK was tested against a panel of 80 binding assays

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encompassing common targets found in the CNS (LeadProfilingScreen2 Panel, Eurofins Scientific).

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Radiochemistry. Radiosynthesis of [18F]TRACK17.

18F-Fluoride

(16220 MBq) was trapped on a QMA light Carb

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cartridge, dried with 2 mL n-butanol and air and eluted using Et4NHCO3 (0.02 M in n-butanol) in reversed

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direction (2×150 µL). The precursor17 (4.8 mg) and Cu(py)2(OTf)2 (5.2 mg) in DMA (400 µL) were added.

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The mixture was stirred at 110°C for 20 min in a heating block with a cap loosely placed on the vial. The

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crude mixture was diluted with 2 mL HPLC solvent (MeCN 55%, H2O 45%) and injected into the HPLC loop

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through a Nylon filter. Semipreparative HPLC was done using a Luna PFP(2) 250 x 4.6 column (MeCN

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55%, H2O 45%, 3 mL/min). The product HPLC fraction was diluted with 20 mL H2O and trapped on a

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SepPak C18 Plus Light cartridge preconditioned with 5 mL EtOH and 10 mL H2O. The cartridge was rinsed

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with 5 mL H2O. The product was eluted with 750 µL EtOH and filtered through a MiniSart SRP 15 filter into

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a sterile vial. The filter was washed with 250 µL EtOH and the eluate was diluted with 9 mL PBS (0.6 M)

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through a Cathivex filter. The product (654 MBq) was obtained in a RCY of 4% and a synthesis time of 84

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min. The effective molar activity was 188 GBq /µmol.

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Preclinical PET Imaging.

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Mouse studies were carried out according to the guidelines of the Canadian Council on Animal Care

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(CCAC) and approved by the local Cross Cancer Institute Animal-Care Committee. Dynamic PET

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experiments of [18F]TRACK were performed on an INVEON PET scanner (Siemens Preclinical Solutions,

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Knoxville, TN, U.S.A.). Female B6129SF2/J mice (The Jackson Laboratory, Bar Harbor, ME, U.S.A.) were


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anesthetized with isoflurane 1.5 Vol% (1 L/min 60% N2 / 40% O2), and body temperature was kept constant

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at 37 °C. Mice were positioned in a prone position into the center of the field of view. No transmission scan

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for attenuation correction was acquired. Mice were injected with 5-8 MBq [18F]TRACK in 100-150 μL saline

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solution containing 8-10% EtOH through a tail vein catheter. Data acquisition was performed over 120 min

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in 3D list mode. The dynamic list mode data were sorted into sinograms with 61 time frames (10 × 2, 8 × 5,

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6 × 10, 6 × 20, 8 × 60, 10 × 120, 13 × 300 s). The frames were reconstructed using maximum a posteriori

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(MAP) as reconstruction mode. No correction for partial volume effects was applied. The image files were

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processed using the ROVER v2.0.51 software (ABX GmbH, Radeberg, Germany). Masks defining 3D

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regions of interest (ROI) were set over the whole mouse brain, and the ROIs were defined by 50%

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thresholding. Mean standardized uptake values [SUVmean = (activity/mL tissue)/(injected activity/body

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weight), mL/g] were calculated for each ROI, and time−activity curves (TACs) were generated. Graphs

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were constructed with GraphPad Prism 5.04 (GraphPad Prism Software Inc., San Diego, CA, U.S.A.). All

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semi-quantified mouse PET data are shown as means ± SEM.

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Human Brain PET Study

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Clinical PET Imaging

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Dynamic PET acquisition on a Siemens Biograph 64 True Point PET/CT scanning device (Siemens

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Medical Solutions, Erlangen, Germany) started simultaneously with the injection of 185 MBq of [18F]TRACK.

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The 90 minutes list mode data were reconstructed with 3D ordered subsets expectation maximization

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(OSEM) algorithm with 4 iterations and 8 subsets and a post-reconstruction Gaussian filter (5 mm FWHM).

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Standard corrections for attenuation, scatter, decay and random counts were performed during image

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reconstruction. The chosen matrix size was 256 × 256 × 109 and the respective voxel size 1.336 × 1.336

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× 2.027 mm (zoom 2). The framing of dynamic PET data was chosen as follows: 12 × 10, 4 × 30, 2 × 60, 2

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× 120, 10 × 300 s. Frame-by-frame motion correction was performed within the PMOD Fusion tool (v3.5,

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PMOD Technologies, Zurich, Switzerland). Additionally, in order to enable a direct visual and quantitative

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comparison with [11C]-(R)-IPMICF16 the previously published dynamic PET data (60 minutes) of the same

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subject were included.16 Image reconstruction and post-processing was the same for both compounds.



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MRI

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For MRI-based definition of anatomical brain regions additionally a T1 MP-RAGE MR image was

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recorded. The three Tesla scanning device (Magnetom Trio, Siemens, Erlangen) was equipped with a 32-

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channel head coil.

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Blood Analysis

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For determination of plasma-to-blood ratio and metabolite analysis four blood samples were taken

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at 16, 30, 61 and 87 min p.i.. Plasma was separated from cells with a centrifugation speed of 3000 × g for

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3 minutes. Whole blood, and whole plasma activity-concentration were measured with a gamma-counter

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(Packard Cobra Quantum).

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For metabolite analysis 300 µL plasma were mixed with 300 µL EtOH to precipitate plasma

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proteins. The mixture was centrifuged at 13500 × g for 1 min and 100 µL supernatant were mixed vigorously

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with each 400 µL n-octanol and PBS (0.06M, pH 7.2). The biphasic mixture was centrifuged at 13500 × g

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for 1 min. 300 µL of each layer were measured in the gamma-counter. The tracer distribution in n-octanol

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and PBS was used as a reference.

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Image Processing

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Standardized uptake value (SUV) images were generated by normalizing with the injected activity

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per subject mass. The T1-weighted MR image was utilized for the definition of anatomical VOIs within

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PMOD Neuro tool (v3.5 PMOD Technologies, Zurich, Switzerland). This procedure is based on the

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maximum probability Hammers atlas (Hammers N30R83)22. A default threshold of 0.3 was applied for gray

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matter (GM) parcellation. Mean SUV data were extracted from static 0-10, 10-30, 30-60 min p.i. images for

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cortical regions, white matter, cerebellar GM and thalamus. Moreover, for relative quantification SUV values

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were divided by white matter uptake, yielding SUV-ratios (SUVR = SUV / SUVWM).


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ASSOCIATED CONTENT

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Supporting Information: Supplementary Table S1: [18F]TRACK quality control results for human use,

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Supplementary Figure S1: PET images mouse brain



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AUTHOR INFORMATION

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Corresponding Author

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Email: [email protected], Phone: 780-248-1829

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Author contributions

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Author contributions: P.B., P. R. N., V.B.-G., A.T., C. W., B. W., D.R. C., R.S. designed research; J.J.B., M.W., S.L., L.K., B.W., P.B., V.B.-G., M.U., J.P.S., R.S. performed research; P.B., R.S. contributed new reagents/analytical tools; J.J.B., M.W., S.L., L.K., B.W., P.B., V.B.-G., R.S. analyzed data; J.J.B., V.B.-G., L.K, R.S wrote the paper.

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Funding

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This work was financially supported by Canada Foundation for Innovation (CFI) project no. 203639 to

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R.S, Cancer Research Society and C17 Council to RS, Natural Science and Engineering Research

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Council of Canada (NSERC) to RS and Weston Brain Institute to RS.

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Note: The authors declare no competing financial interest.

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V.B.-G.

and R.S. contributed equally to this work.

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ACKNOWLEDGEMENT

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TABLE

Table 1. Screening of Inhibitor [19F]TRACK for CNS off-target bindinga

Target

% Inhibition of Control Specific Binding

Target

% Inhibition of Control Specific Binding

5-HTT (SERT) (h)

-7.79

ETA (h)

3.46

5-HT1A (h)

2.38

ETB (h)

4.84

5-HT1B

-21.40

GABA

-10.20

5-HT2A (h)

3.22

GAL1 (h)

-30.60

5-HT2B (h)

0.72

GAL2 (h)

-0.75

5-HT2C (h

-3.22

GR (h)

-6.01

5-HT3 (h

1.55

H1 (h)

1.11

5-HT5a (h)

2.12

H2 (h)

-8.74

5-HT6 (h)

6.72

IP (PGI2) (h)

-15.90

5-HT7 (h)

2.02

kappa (KOP)

-3.82

A1 (h)

25.22

KV channel

-5.85

A2A (h)

-5.42

M1 (h)

-10.10

A3 (h)

11.51

M2 (h)

-6.96

1

-1.60

M3 (h)

-3.93

2

-5.78

M4 (h)

4.37

AT1 (h)

-4.41

M5 (h)

-7.81

AT2 (h)

-1.12

MC4 (h)

-3.97

B2 (h)

5.24

MT1 (ML1A) (h)

-0.96

BB

-6.72

mu (MOP) (h)

-1.01

1 (h)

5.92

Na+ channel (site 2)

-10.20

2 (h)

-3.29

NK1 (h)

-3.89

0

NK2 (h)

-9.30

BZD (peripheral)

-0.28

NK3 (h)

-0.82

Ca2+ channel (L, verapamil site)

-3.97

NOP (ORL1) (h)

-5.12

CB1 (h)

4.00

norepinephrine transporter (h)

-3.96

CCK1 (CCKA) (h)

4.40

NTS1 (NT1) (h)

-10.00

CCK2 (CCKB) (h)

-11.20

P2X

-7.96

CCR1 (h)

10.71

P2Y

6.59

BZD (central)



Page 20 of 27

CGRP (h)

-4.79

PAC1 (PACAP) (h)

-0.13

Cl- channel (GABA-gated)

6.30

PCP

-6.12

CXCR2 (IL-8B) (h)

-9.99

PDGF

6.33

D1 (h)

-3.35

PPAR- (h)

-2.02

D2S (h)

4.69

sigma (h)

3.67

D3 (h)

5.77

SKCa channel

-5.95

D4.4 (h)

-5.60

sst (h)

-11.70

D5 (h)

3.00

TNF-alpha (h)

5.90

delta (DOP) (h)

-1.10

V1a (h)

2.12

dopamine transporter (h)

6.91

VPAC1 (VIP1) (h)

-2.74

EP2 (h)

14.86

dopamine transporter (h)

6.00

EP4 (h)

-2.20

5-HT transporter (h)

3.29

a5-HTT,

serotonin transporter; 5-HT, 5-hydroxytryptamine receptors; A, adenosine receptor;  alpha adrenergic receptor; AT, Angiotensin II receptor; BB, bombesin receptors;  beta adrenergic receptor; BZD, benzodiazepine receptors; CB1, cannabinoid receptor type 1; CCK, cholecystokinin receptors; CCR1, C-C chemokine receptor type 1; CGRP, calcitonin gene-related peptide receptor; CXCR2, C-X-C chemokine receptor type 2; D, dopamine receptor; OP, opioid receptor; EP, Prostaglandin receptor; ET, endothelin receptors; GABA, gamma-aminobutyric acid; GAL, galanin receptor; GR, glucocorticoid receptor; H, histamine receptor; IP, prostacyclin receptor; KV, voltage-gated K+ channels; M, muscarinic acetylcholine receptor; MC4, melanocortin 4 receptor; NK, tachykinin receptor; NTS1, neurotensin receptor 1; P2, purinergic receptor; PAC1, pituitary adenylate cyclase-activating polypeptide type I receptor; PDGF, platelet-derived growth factor receptor; PPAR-, peroxisome proliferator-activated receptor gamma; SK, small conductance calcium-activated potassium channels; sst, somatostatin receptor; TNF, tumor necrosis factor alpha; V1a, vasopressin receptor 1A; VPAC1, vasoactive intestinal polypeptide receptor 1.


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Table 2. SUV-ratios

[18F]TRACK Thalamus Cerebellum White matter Cortex

[11C]-(R)-IPMICF16

0-10

10-30

30-60

60-90

0-10

10-30

30-60

1.21 1.45 1.00 1.24

1.25 1.33 1.00 1.20

1.23 1.23 1.00 1.11

1.16 1.11 1.00 1.01

1.27 1.30 1.00 1.16

1.20 1.20 1.00 1.12

1.19 1.12 1.00 1.07



FIGURES LEGENDS

Figure 1. Selected clinical pyrazolo[1,5a]pyrimidine-imidazo[1,2-b]pyridazine-based pan-Trk inhibitors and Trk-targeted PET radioligands.

Figure 2. Unmetabolized fraction of [18F]TRACK in plasma.

Figure 3. In vivo PET imaging of [18F]TRACK (a) and [11C]-(R)-IPMICF16 (b) in the human brain. Top row: T1MP-RAGE MR image of the subject. Bottom rows: overlays of the 0-10, 10-30, 30-60 and 60-90 min p.i. summed SUV PET images with the MR image.

Figure 4. Time-activity curves (TACs) from thalamus, cerebellar GM, cortical GM and white matter of [18F]TRACK (a) and [11C]-(R)-IPMICF16 (b) in the human brain. The TACs have been cut at 0.8 SUV for clarity (SUVpeak values range from 1.15 in the white matter to 2.3 at ~30 s post-injection).


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FIGURES

Figure 1. Selected clinical pyrazolo[1,5a]pyrimidine-imidazo[1,2-b]pyridazine-based pan-Trk inhibitors and Trk-targeted PET radioligands.



Figure 2. Unmetabolized fraction of [18F]TRACK in plasma.


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Figure 3. In vivo PET imaging of [18F]TRACK (a) and [11C]-(R)-IPMICF16 (b) in the human brain. Top row: T1MP-RAGE MR images of the subject. Bottom rows: overlays of the 0-10, 10-30, 30-60 and 60-90 min p.i. summed SUV PET images with the MR images. Scans with both radioligands were performed in the same healthy human subject.



b)

a) 0.8

Tissue activity (SUV)

Tissue activity (SUV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

0.7 0.6 0.5 0.4 0.3 0

20 40 60 Time (min)

80

0.8 0.7

Thalamus

0.6

Cerebellar GM

0.5

Cortex WM

0.4 0.3 0

20

40 60 Time (min)

80

Figure 4. Time-activity curves (TACs) from thalamus, cerebellar GM, cortical GM and white matter of [18F]TRACK (a) and [11C]-(R)-IPMICF16 (b) in the human brain. The TACs have been cut at 0.8 SUV for clarity (SUVpeak values range from 1.15 in the white matter to 2.3 at ~30 s post-injection).


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GRAPHICAL TABLE OF CONTENT


First-in-Human Brain Imaging of [18F]TRACK, a PET tracer for

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